Abstract
Numerous cellular processes are regulated by microRNAs (miRNAs), both cellular and viral. Elucidating the targets of miRNAs has become an active area of research. An important method in this field is cross-linking and immunoprecipitation (CLIP), where cultured cells or tissues are UV-irradiated to cross-link protein and nucleic acid, the RNA binding protein of interest is immunoprecipitated, and the RNAs pulled down with the protein are isolated, reverse-transcribed, and analyzed by sequencing. CLIP using antibody against Argonaute (Ago), which binds to both miRNA and mRNA as they interact in RISC, has allowed researchers to uncover a large number of miRNA targets. Coupled with high-throughput sequencing, CLIP has been useful for revealing miRNA targetomes for the γ-herpesviruses Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Variants on the CLIP protocol are described, with the benefits and drawbacks of each. In particular, the most recent methods involving RNA–RNA ligation to join the miRNA and its RNA target have aided in target identification. Lastly, data supporting biologically meaningful interactions between miRNAs and long non-coding RNAs (lncRNAs) are reviewed. In summary, ribonomics-based miRNA targetome analysis has expanded our understanding of miRNA targeting and has provided a rich resource for EBV and KSHV research with respect to pathogenesis and tumorigenesis.
Highlights
MicroRNAs are short, non-coding RNAs of 21–23 nt that bind to target RNAs, inhibiting translation or inducing degradation or both [1]
Ribonomics data accelerates our understanding of miRNAs in viral cancer: The rapid development and evolution of innovative ribonomics approaches to determine global miRNA targetomes has enabled study of how miRNAs, and here miRNAs expressed by the tumorigenic γ-herpesviruses Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV), shape host cell transcriptomes
Several detailed molecular studies have already arisen from target catalogs created by high-throughput sequencing (HITS)-cross-linking and immunoprecipitation (CLIP) and PAR-CLIP datasets and new ones will follow utilizing qCLASH datasets that have been generated for KSHV and are in progress for additional herpesviruses
Summary
MicroRNAs (miRNAs) are short, non-coding RNAs of 21–23 nt that bind to target RNAs, inhibiting translation or inducing degradation or both [1]. One strand becomes incorporated into RISC, the RNA-induced silencing complex, where it interacts with the target transcript to enable silencing [2]. The importance of miRNAs in cancer is underlined by the fact that several KSHV and EBV miRNAs have roles in processes associated with tumorigenesis [5,6]. Investigation of interactions between miRNAs and RISC-associated RNAs with ribonomics methods that do not rely upon assumptions about seed sequences have uncovered the existence of many long non-coding RNAs (lncRNAs) as potential miRNA targets [12]. In a few examples, miRNAs clearly can regulate lncRNA levels and appear to play roles in important biological processes such as regulation of cell cycle, metastasis, and stem cell differentiation [13]
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